Characterization of muscarinic receptors mediating release of epithelial derived relaxant factor (EpDRF) in guinea-pig isolated trachea

Summary Muscarinic receptors mediating the release of epithelial derived relaxant factor (EpDRF) have been studied by using both contractions of the guinea-pig tracheal strip (with epithelium intact or denuded) or a coaxial bioassay assembly (rat anococcygeus-recipient; guinea-pig trachea-donor tissue). Indomethacin (1 M/l) and physostigmine (0.1 M/l) were both present throughout the study.In the tracheal strip studies, the potencies and maximal effects of all agonists studied (acetylcholine, arecoline, bethanechol, carbachol, (+)cis-dioxolane, ethoxyethyltrimethylammonium, L-660,863, (±)methacholine and OXA-22) were not affected or were only slightly (but significantly) reduced by removal of the epithelium. The -log K_B for the muscarinic antagonists, atropine, pirenzepine, methoctramine and 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine) were also not affected and the -log K_B values were consistent with M₃ muscarinic receptor function. However, the -log K_B value of para-fluoro-hexahydro-siladifendol (p-F-HHSiD) was significantly (P < 0.05) increased upon epithelial denudation (epithelium intact, 7.1; epithelium removed, 7.6). The coaxial bioassay assembly provided more convincing evidence for release of EpDRF in that all muscarinic agonists studied caused relaxations of a precontracted anococcygeus tissue. These relaxations were observed only in the presence of a tracheal tube possessing an intact epithelium. The rank order of potencies for agonists at receptors mediating EpDRF dependent relaxation were similar to those estimated at receptors causing contraction. These data suggested that a substantial receptor reserve was associated with the receptors mediating both EpDRF release and contraction. The affinities of the muscarinic antagonists (atropine, pirenzepine, methoctramine, p-F-HHSiD, 4-DAMP and gallamine) indicated that M₃ receptors also mediated EpDRF release.It is concluded that EpDRF release in guinea-pig trachea is a general property of muscarinic agonists and that this process is mediated, like the contractile response, by M₃ receptors. Send offprint requests to R. M. Eglen at the above address     

Muscarine receptor types mediating autoinhibition of acetylcholine release and sphincter contraction in the guinea-pig iris

Summary The potencies of several muscarine receptor antagonists in blocking either the autoinhibition of acetylcholine release or the muscarinic contraction of the sphincter muscle upon acetylcholine release were investigated in the guinea-pig iris. The agonist at pre- or postjunctional muscarine receptors was acetylcholine released upon field stimulation (5.5 Hz, 2 min) of the irides preloaded with ¹⁴C-choline. The stimulation-evoked ¹⁴C-overflow was doubled in the presence of atropine 0.1 mol/l but unaffected by the agonist (±)-methacholine (50 mol/l). Thus, under the present stimulation conditions, the autoinhibition of acetylcholine release on the guinea-pig iris cholinergic nerves was nearly maximally activated. Isotonic contractions of the irides upon field stimulation consisted of a rapid, atropine (0.1 mol/l). peak phase followed by a sustained contraction which involved a cholinergic and a non-cholinergic stimulation of the sphincter muscle. The M₂-selective antagonists methoctramine (10 mol/l) and gallamine (100 µmol/l). increased both the ¹⁴Goverflow and the peak contractions evoked by field stimulation. In contrast, the M₃-selective antagonist hexahydrosiladifenidol (0.1–10 mol/l) failed to affect the evoked ¹⁴C-release but concentration-dependently (1–10 mol/l) reduced the iris contractions. Pirenzepine (10 mol/l) enhanced the evoked ¹⁴C-overflow and inhibited the peak contractions (0.1–10 mol/l; maximal effect at 10 mol/l). The low potency of the antagonist at both receptor sites indicates that an M₁ muscarine receptor is not involved. The results are consistent with the idea of M₂ muscarine receptors mediating autoinhibition of acetylcholine release in the guinea-pig iris and M₃-like receptors inducing the contraction of the sphincter muscle. Send offprint requests to I. T. Bognar at the above address     

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

1. The endothelin (ET) receptor subtype that mediates niric oxide (NO)-dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea-pig trachea using a recently developed porphyrinic microsensor was also measured.
2. ET-1 (1 pM–100 nM) contracted tracheal tube preparations pretreated with the NO-synthase inhibitor, L-NMMA, and relaxed, in an epithelium-dependent manner, preparations pretreated with the inactive enantiomer D-NMMA. The effect of L-NMMA was reversed by L-Arg, but not by D-Arg.
3. The selective ET_B receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM–100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D-NMMA or with L-NMMA. In the presence of the ET_A receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), ET-1 administration resulted in a contraction that was similar after either L-NMMA or D-NMMA. In the presence of the ET_B receptor antagonist, BQ788 (1 μM), ET-1 relaxed and contracted tracheas pretreated with D-NMMA and L-NMMA, respectively.
4. Exposure of tracheal segments to ET-1 (1–1000 nM) caused a concentration-dependent increase in NO release that was reduced by L-NMMA. IRL1620 (1 μM) did not cause any significant NO release. {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (10 μM), but not, BQ788 (1 μM), inhibited the NO release induced by ET-1.
5. These results demonstrate that in the isolated guinea-pig trachea activation of ET_B receptors results in a contractile response, whereas activation of ET_A receptors cause both a contraction, and an epithelium-dependent relaxation that is mediated by NO release.

     

Differences in the prejunctional effects of methacholine and pilocarpine on the release of endogenous acetylcholine from guinea-pig trachea

We investigated the effects of the full muscarinic acetylcholine receptor agonist methacholine and the partial and putatively M₂-selective agonist pilocarpine on endogenous acetylcholine release from guinea-pig trachea by use of high-performance liquid chromatography with electrochemical detection. Atropine-induced increases in acetylcholine release were used to monitor the system.Electrical field stimulation (8 V, 30 Hz, 0.5 ms for 5 min)-induced acetylcholine release in the presence of neostigmine, with or without preincubation with choline to maximally enhance acetylcholine output, was increased to about 225% by 0.3 M atropine, indicating functional autoinhibition. However, methacholine (10 M) did not affect the acetylcholine release, whereas it was enhanced to 166% by 30 M pilocarpine. When electrical field stimulation was applied at lower intensity (8 V, 16 Hz, 0.1 ms for 5 min) and in the absence of neostigmine, an increase by 0.3 M atropine (to 177%) but a decrease of the acetylcholine release by 10 M methacholine (to 65%) and 30 M pilocarpine (to 63%) were observed. These results clearly demonstrate (i) that inhibition of evoked endogenous acetylcholine release from prejunctional nerve terminals in guinea-pig trachea can only be demonstrated under conditions of low junctional concentrations of acetylcholine, and (ii) that pilocarpine, as a partial muscarinic agonist, behaves as an antagonist under high junctional concentrations of the neurotransmitter.     

Beta-adrenoceptors invloved in inhibition of histamine release from sensitized guinea-pig lung.

Selective β-adrenoceptor agonists and antagonists were used to study the type of β-adrenoceptors involved in inhibiting antigen-induced histamine release from actively sensitized guinea-pig lung. Results obtained with six non-catechol β-adrenergic agonists were compared with those found in guinea-pig atrial (β₁) and tracheal (β₂) preparations. In terms of rank order the relative activities of the compounds differed in the three preparations. Dissociation constants (K_B values) for the cardioselective antagonist H93/26 were assessed using (?)-isoprenaline as an agonist. The K_B value for inhibition of histamine release was significantly different from, and intermediate between, the K_B values obtained in atria and trachea. In the guinea-pig tissues H35/25 was not a selective β-adrenoceptor antagonist; K_B values were not significantly different in the three preparations. The results using the β-adrenoceptor agonists and antagonists suggest that the β-receptors involved in inhibition of antigen-induced histamine release in the guinea-pig lung differ from those found in guinea-pig atria and trachea.     

Characterization of the prejunctional muscarinic receptors mediating inhibition of evoked release of endogenous noradrenaline in rabbit isolated vas deferens

The aim of the present study was to characterize the prejunctional modulation of evoked release of endogenous noradrenaline in rabbit vas deferens by the use of muscarinic receptor agonists and subtype-prefering antagonists.Vasa deferentia of the rabbit were stimulated electrically by trains of 120 pulses delivered at 4 Hz or trains of 30 pulses at 1 Hz. The inhibition by muscarinic agonists of the stimulation-evoked overflow of endogenous noradrenaline in the absence and presence of antagonists was used to determine affinity constants for antagonists. These values were compared with those observed at putative M₁ receptors inhibiting neurogenic twitch contractions in the rabbit vas deferens and with affinity data obtained at M₁(m₁)-M₄(m₅) receptors in functional studies and binding experiments.The evoked overflow of noradrenaline from sympathetic nerves was enhanced by the Al receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the P₂ purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS) and indomethacin, indicating a tonic inhibition by endogenous A₁ and P₂ purinoceptor agonists and prostanoids, respectively. The stimulation-evoked overflow at 4 Hz was not sensitive to inhibition by the muscarinic agonists methacholine or 4-(4-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343). In contrast, at a stimulation frequency of 1 Hz the evoked noradrenaline release was decreased by muscarinic agonists (EC₅₀): arecaidine propargyl ester (0.062 M), 4-Cl-McN-A-343 (0.32 M), 4-(4-fluorophenylcarbamoyloxy)-2-butynylN-methyl-pyrrolidinium tosylate (4-F-PyMcN⁺; 0.48 M) and methacholine (0.86 M). The affinity constants of most of the muscarinic antagonists [atropine: pK_B = 9.47; (R)-trihexyphenidyl: pK_B = 9.18; pirenzepine: pA₂ = 7.68; methoctramine: pK_B = 6.90] are consistent with estimates of these antagonists at M₁(m₁) receptors determined in various functional and binding studies. The high antagonistic potency of pirenzepine and (R)-trihexyphenidyl and the agonistic activity of 4-F-PyMcN⁺ argue for the involvement of M₁, and against that of M₂ and M₃ receptors in the inhibition of evoked noradrenaline overflow. However, the high apparent pK_B of 8.30 for himbacine is not in accordance with an M₁ receptor; by contrast, it would be compatible with the presence of M₂ or M₄ receptors. The potencies of the tested muscarinic agonists and antagonists largely agree with those obtained for the inhibition of neurogenic twitch responses (0.05 Hz) in the rabbit vas deferens. In conclusion, the rabbit vas deferens is endowed with prejunctional muscarinic receptors mediating heteroinhibition of noradrenaline release that are probably of the same subtype as the putative M₁ receptors inhibiting neurogenic twitch contractions, and are not of the M₂, M₃ or m₅ subtype. Correspondence to: U. Grimm at the above address     

Inhibitory and excitatory muscarinic receptors modulating the release of acetylcholine from the postganglionic parasympathetic neuron of the chicken heart

Summary The effects of muscarinic receptor antagonists on ACh release were studied in the absence or presence of cholinesterase (ChE) inhibition using the isolated perfused chicken heart. Presynaptic inhibitory muscarinic autoreceptor were characterized by determining the potency of various antagonists to enhance [³H]-ACh release evoked by field stimulation (3 Hz, 1 min). The order of potencies was: (±)-telenzepine > atropine > 4-DAMP > silahexocyclium > pirenzepine > hexahydro-siladifenidol > AF-DX 116. The comparison with known pA₂ values for M₁-, M₂- and M₃-receptors revealed that the presynaptic autoreceptor meets the criteria of an M₁-receptor. Basal, not electrically evoked overflow of unlabelled ACh into the perfusate was caused by leakage release (non-exocytotic), as it was independent of extracellular Ca²⁺ . Muscarinic receptor antagonists failed to enhance basel overflow. In contrast, when ChE activity was inhibited by 10^–6M tacrine or pretreatment with 10^–4M DFP, the ACh overflow was partially Ca²⁺-dependent and was reduced by tetrodotoxine. Moreover, block of the inhibitory muscarinic autoreceptors by (±)-telenzepine or pirenzepine caused a several-fold enhancement of the ACh release. The potencies of these antagonists were identical to those found for the electrically evoked [³H]-ACh release. The rate of ACh release enhanced by ChE inhibition plus telenzepine corresponds to about 12% of the total ACh pool per min, which is about the maximum amount of ACh that is available for any kind of stimuli. The release was dependent on the presence of exogenous choline. Hence elevation of ACh release led to a correspondingly enhanced ACh synthesis. The dramatic enhancement of ACh release by the ChE inhibition in combination with a block of presynaptic muscarinic autoinhibition was not inhibited by (+)-tubocurarine but by atropine (10^–9 to 10^–7 M) or 10^–6 M telenzepine. It is concluded that basal release of ACh in the heart was due to non-exocytotic leakage release. Inhibition of ChE led to a marked stimulation of excitatory muscarinic receptors of the intrinsic parasympathetic neuron with a consecutive postganglionic release of ACh. The strong postganglionic excitation was obvious when the inhibitory muscarinic autoreceptors were selectively blocked. Of the two described muscarinic receptors found in the parasympathetic postganglionic neuron of the chicken heart only the inhibitory was classified as being M₁, whereas the subtype of the excitatory one is unlike M₁ and remains to be identified.Preliminary results have been presented at the Spring meeting of the German Pharmacological Society in 1992 (Brehm and Lindmar 1992) Correspondence to R. Lindmar at the above address     

The detection of the non-M2 muscarinic receptor subtype in the rat heart atria and ventricles

Mammal heart tissue has long been assumed to be the exclusive domain of the M(2) subtype of muscarinic receptor, but data supporting the presence of other subtypes also exist. We have tested the hypothesis that muscarinic receptors other than the M(2) subtype are present in the heart as minor populations. We used several approaches: a set of competition binding experiments with pirenzepine, AFDX-116, 4-DAMP, PD 102807, p-F-HHSiD, AQ-RA 741, DAU 5884, methoctramine and tripinamide, blockage of M(1) muscarinic receptors using MT7 toxin, subtype-specific immunoprecipitation experiments and determination of phospholipase C activity. We also attempted to block M(1)-M(4) receptors using co-treatment with MT7 and AQ-RA 741. Our results show that only the M(2) subtype is present in the atria. In the ventricles, however, we were able to determine that 20% (on average) of the muscarinic receptors were subtypes other than M(2), with the majority of these belonging to the M(1) subtype. We were also able to detect a marginal fraction (6 +/- 2%) of receptors that, based on other findings, belong mainly to the M(5) muscarinic receptors. Co-treatment with MT7 and AQ-RA 741 was not a suitable tool for blocking of M(1)-M(4) receptors and can not therefore be used as a method for M(5) muscarinic receptor detection in substitution to crude venom. These results provide further evidence of the expression of the M(1) muscarinic receptor subtype in the rat heart and also show that the heart contains at least one other, albeit minor, muscarinic receptor population, which most likely belongs to the M(5) muscarinic receptors but not to that of the M(3) receptors.     

Presynaptic muscarinic receptor subtypes involved in the inhibition of acetylcholine and noradrenaline release in bovine cerebral arteries

Summary Experiments were performed in bovine cerebral arteries preincubated with [³H]-choline or [³H]-noradrenaline to analyze the presynaptic muscarinic receptors involved in inhibition of acetylcholine and noradrenaline release induced by electrical stimulation (4 Hz, 200 mA, 0.3 ms, 1 min). For this purpose, the actions of several muscarinic receptor antagonists on the ³H overflow and on the carbacol-induced inhibition of this overflow were assessed. The evoked [³H]-acetylcholine release and [³H]-noradrenaline release were markedly reduced by the presence of tetrodotoxin, Ca²⁺-free medium, and the inhibitor of both choline transport and choline acetyltransferase, AF64A. Chemical sympathetic denervation with 6-hydroxydopamine (6-OHDA) decreased the uptake of[³H]-noradrenaline, and AF64A reduced mainly the uptake of [³H]-choline, but also of [³H]-noradrenaline. Carbachol reduced the evoked [³H]-noradrenaline and [³H]-acetylcholine release; the IC₅₀ values were 0.37 and 0.43 mol/l, respectively.Atropine and 4-DAMP, but not AF DX 116, methoctramine or pirenzepine, increased the evoked [³H]-acetylcholine release. However, these muscarinic antagonists failed to modify the evoked [³H]-noradrenaline release. Carbachol inhibited the release of both acetylcholine and noradrenaline. The inhibition was blocked by the antagonists. The rank orders of potency (based on plC₅₀ values) were, in the case of [³H]-acetylcholine release, atropine > 4-DAMP >AF-DX 116 >- pirenzepine >- methoctramine, and, in the case of [³H]-noradrenaline release, atropine > 4-DAMP > AF-DX 116 >- methoctramine >-pirenzepine. These results suggest (1) that the prosynaptic receptors that modulate endogenous acetylcholine release are likely of the M₃ subtype, whilst those involved on the effect of the exogenous agonist Carbachol are of M₂ subtype, and (2) that those which inhibit noradrenaline release are probably a mixture of M₂ and M₃ subtypes as well. The autoinhibition of the acetylcholine release was funtionally active under our experimental conditions, while noradrenaline release does not appear to be modulated by muscarinic receptors in physiological conditions.Send offprint requests to G. Balfagón at the above address     

The isolated lung strip and single open tracheal ring: a convenient combination for characterizing Schultz Dale anaphylactic contractions in the peripheral and central airways of the guinea-pig

1. The use of the isolated lung strip and single open tracheal ring for studying Schultz Dale anaphylactic contractions in both the peripheral and central airways is described. 2. This method is particularly relevant to studies of anaphylaxis because many preparations may be obtained from a single sensitized animal. 3. Isolated preparations from control guinea-pigs did not respond to ovalbumin, whereas the lung strip and trachea taken from guinea-pigs sensitized to ovalbumin, contracted markedly to that antigen. 4. The peripheral and central airways from sensitized animals responded in the same way to antigen challenge.     

知母皂苷元及其异构体对老年大鼠学习记忆和脑内M_1受体密度的影响

目的　观察知母皂苷元 (ZMS)及其异构体 (ZMR)对老年大鼠学习记忆和脑内M1受体密度的作用。方法　选择 2 4mon龄SD老年大鼠 ,将动物分为老年对照组、ZMS组和ZMR组 ,并以 3～ 4mon龄青年大鼠作为正常对照 ,用迷宫法测定学习记忆能力 ,采用放射配基结合分析法测定脑内M1受体密度。结果　用药组大鼠连续口服ZMS和ZMR 4 0d后 ,与老年对照组比较 ,其学习记忆能力明显增强 ,脑内M1受体密度升高。结论　知母皂苷元及其异构体对老年性痴呆的胆碱能系统功能渐进性退化有一定的预防和治疗作用     

Novel selective cannabinoid CB(1) receptor antagonist MJ08 with potent in vivo bioactivity and inverse agonistic effects

Aim:

To characterize the biological profiles of MJ08, a novel selective CB₁ receptor antagonist.

Methods:

Radioligand binding assays were performed using rat brain and spleen membrane preparations. CB₁ and CB₂ receptor redistribution and intracellular Ca²⁺ ([Ca²⁺]_i) assays were performed with IN CELL Analyzer. Inverse agonism was studied using intracellular cAMP assays, and in guinea-pig ileum and mouse vas deferens smooth muscle preparations. In vivo pharmacologic profile was assessed in diet-induced obesity (DIO) mice.

Results:

In radioligand binding assay, MJ08 selectively antagonized CB₁ receptor (IC₅₀=99.9 nmol/L). In EGFP-CB₁_U2OS cells, its IC₅₀ value against CB₁ receptor activation was 30.23 nmol/L (SR141716A: 32.16 nmol/L). WIN 55,212-2 (1 μmol/L) increased [Ca²⁺]_i in the primary cultured hippocampal neuronal cells and decreased cAMP accumulation in CHO-hCB₁ cells. MJ08 (10 nmol/L–10 μmol/L) blocked both the WIN 55,212-2-induced effects. Furthermore, MJ08 reversed the inhibition of electrically evoked twitches of mouse vas deferens by WIN 55,212-2 (pA₂=10.29±1.05). MJ08 and SR141716A both showed an inverse agonism activity by markedly promoting the contraction force and frequency of guinea pig ileum muscle. MJ08 significantly increased the cAMP level in CHO-hCB₁ cells with an EC₅₀ value of 78.6 nmol/L, which was lower than the EC₅₀ value for SR141716A (159.2 nmol/L). Besides the more potent pharmacological effects of cannabinoid CB₁ receptor antagonism in DIO mice, such as reducing food intake, decreasing body weight, and ameliorating dyslipidemia, MJ08 (10 mg/kg) unexpectedly raised the fasted blood glucose in vivo.

Conclusion:

MJ08 is a novel, potent and selective CB₁ receptor antagonist/inverse agonist with potent bioactive responses in vitro and in vivo that may be useful for disclosure the versatile nature of CB₁ receptors.     

Pharmacological characterization of the first potent and selective antagonist at the cysteinyl leukotriene 2 (CysLT2) receptor

Background and purpose:

Cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory and cardiovascular disorders. Their actions are mediated by CysLT₁ and CysLT₂ receptors. Here we report the discovery of 3-({[(1S,3S)-3-carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy) benzoic acid (HAMI3379), the first potent and selective CysLT₂ receptor antagonist.

Experimental approach:

Pharmacological characterization of HAMI3379 was performed using stably transfected CysLT₁ and CysLT₂ receptor cell lines, and isolated, Langendorff-perfused, guinea pig hearts.

Key results:

In a CysLT₂ receptor reporter cell line, HAMI3379 antagonized leukotriene D₄- (LTD₄-) and leukotriene C₄- (LTC₄-) induced intracellular calcium mobilization with IC₅₀ values of 3.8 nM and 4.4 nM respectively. In contrast, HAMI3379 exhibited very low potency on a recombinant CysLT₁ receptor cell line (IC₅₀ > 10 000 nM). In addition, HAMI3379 did not exhibit any agonistic activity on both CysLT receptor cell lines. In binding studies using membranes from the CysLT₂ and CysLT₁ receptor cell lines, HAMI3379 inhibited [³H]-LTD₄ binding with IC₅₀ values of 38 nM and >10 000 nM respectively. In isolated Langendorff-perfused guinea pig hearts HAMI3379 concentration-dependently inhibited and reversed the LTC₄-induced perfusion pressure increase and contractility decrease. The selective CysLT₁ receptor antagonist zafirlukast was found to be inactive in this experimental setting.

Conclusions and implications:

HAMI3379 was identified as a potent and selective CysLT₂ receptor antagonist, which was devoid of CysLT receptor agonism. Using this compound, we showed that the cardiac effects of CysLTs are predominantly mediated by the CysLT₂ receptor.     

Effect of the selective 5-HT1A receptor antagonist WAY 100635 on the inhibition of e.p.s.ps produced by 5-HT in the CA1 region of rat hippocampal slices

1. The actions of N-(2-(-4(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), a novel and selective 5-hydroxytryptamine_1A (5-HT_1A) antagonist, on excitatory postsynaptic potentials (e.p.s.ps) were investigated by use of intracellular recordings in pyramidal cells of the CA1 region of rat hippocampal slices.
2. WAY 100635 (10 nM) did not affect any of the investigated parameters of cell excitability such as membrane potential, total input resistance (R_in), firing threshold, action potential amplitude, action potential frequency adaptation, and slow afterhyperpolarization (sAHP) which follows repetitive firing of action potentials. WAY 100635 did not have any effect on either the slope or the amplitude of e.p.s.ps evoked by stimulation of the CA1 stratum radiatum.
3. Bath application of either 5-hydroxytryptamine (5-HT, 10–30 μM) or 5-carboxamidotryptamine (5-CT, 300 nM) hyperpolarized the membrane potential (ΔV_m=−4.1±0.9 and −6.0±0.9 mV, respectively), and reduced R_in (−25±8% and −18±1%, respectively). 5-HT blocked the action potential frequency adaptation and significantly reduced the amplitude of the sAHP that follows repetitive firing of action potentials.
4. 5-HT significantly decreased the amplitude of evoked e.p.s.ps (−14±6%). This effect was greater in the presence of the GABA_A receptor antagonist bicuculline (10 μM, −45±12%) and was mimicked by 5-CT (−49±5%). Both AMPA and NMDA components of e.p.s.ps were significantly reduced in amplitude by 5–HT (−38±8%, n=6, and −29±12%, n=3, respectively; P<0.05).
5. WAY 100635 fully antagonized the hyperpolarization, the reduction of R_in, and the decrease in amplitude of e.p.s.ps elicited by 5-HT, while it did not affect the action of 5-HT on the action potential frequency adaptation. In the presence of WAY 100635, 5-HT elicited a depolarization which was blocked by 10–30 μM RS 23597-190, a selective 5-HT₄ receptor antagonist.
6. Our data demonstrate that WAY 100635 is devoid of direct effects on CA1 pyramidal cell excitability and on evoked e.p.s.ps, while it fully antagonizes the effects of 5-HT on excitatory synaptic transmission and on hyperpolarization, without affecting the 5-HT₄ receptor-mediated response. Since WAY 100635 selectively antagonizes 5-HT_1A receptor-mediated actions of 5-HT, our data also demonstrate that the inhibitory action of 5-HT on excitatory synaptic transmission in CA1 is mediated by 5-HT_1A receptors.

     

Influence of the 5-HT6 receptor on acetylcholine release in the cortex: pharmacological characterization of 4-(2-bromo-6-pyrrolidin-1-ylpyridine-4-sulfonyl)phenylamine,a potent and selective 5-HT6 receptor antagonist

A small series of aryl pyridyl sulfones has been prepared and investigated for its 5-HT(6) receptor binding properties. Thereof, pyrrolidinyl derivative 11 proved to be a very potent (pK(i) 9) and selective 5-HT(6) receptor antagonist. By means of in vivo microdialysis in the frontal cortex and a passive avoidance paradigm, where 11 reversed a scopolamine induced retention deficit, a functional correlation between 5-HT(6) receptors and cholinergic neurotransmission could be shown, supporting the therapeutic potential of 5-HT(6) receptors in the treatment of cognitive deficits.     

Characterization of the molecular interactions of interleukin-8 (CXCL8), growth related oncogen alpha (CXCL1) and a non-peptide antagonist (SB 225002) with the human CXCR2

Neutrophil recruitment to inflammatory sites is mediated by two related receptors: CXC chemokine receptors 1 (CXCR1) and 2 (CXCR2). Both receptors share two ligands, interleukin-8 (CXCL8) and GCP-2 (CXCL6), whereas several chemokines, including growth related oncogen alpha (CXCL1) and a non-peptide antagonist (SB 225002) are specific for CXCR2. The objective of this study was to map the different amino acids involved in the binding and activation/inhibition of human CXCR2. This was performed by exchanging non-conserved amino acids of CXCR2 with their counterparts in CXCR1. The mutants generated showed that: (a) for CXCL8 binding, the N-terminus of CXCR1 and the second extra-cellular loop of CXCR2 are determinant, the N-terminus of CXCR2 is not sufficient and the transmembrane domain seven is probably involved; (b) for CXCL1, the N-terminus of CXCR2 is necessary but not sufficient for binding. The activation study indicated that amino acids critical for activation are not necessarily involved in binding process. Finally, the mechanism of binding of a non-peptide antagonist on CXCR2 was investigated: it occurred through epitopes (a) which were disseminated within the receptor, (b) which differed according to the use of CXCL8 or CXCL1 as a competitor and (c) which did not necessarily overlap with agonist binding sites. We also showed that inhibition of binding and inhibition of activation involved different amino acids.     

Prolonged treatment with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) differently affects the serotonergic modulation of cortical acetylcholine release in male and female guinea pigs

Acetylcholine (ACh) release from the cerebral cortex was studied in freely moving guinea pigs, implanted with epidural cups. A single dose of either 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT, 0.1 mg/kg s.c.) or 5-hydroxytryptamine (5-HT, 250 μg i.c.v.) increased cortical ACh release, both in male and in female guinea pigs. In female guinea pigs chronically treated with 8-OHDPAT (0.1 mg/kg daily s.c., for 14 days) the facilitatory effect of 8-OHDPAT and 5-HT was maintained. In chronically 8-OHDPAT-treated male guinea pigs, 8-OHDPAT no longer modified ACh release, while 5-HT inhibited it. The inhibition was prevented by the 5-HT₃ antagonist MDL 72222, 1 mg/kg s.c. These results indicate that differences exist between male and female guinea pigs in the adaptive responses to prolonged treatment with the selective 5-HT_1A agonist 8-OHDPAT.